Week 15&16 EnzymeAssay
Enzyme assay with stock YopH enzyme was performed.
Figure 1: Results of enzyme assay from 234uM sample of YopH enzyme.
Sample Absorbance at 410nm
A (no enzyme) 0
B (10ng enzyme) 0.054
C (20ng enzyme) 0.422
D (40ng enzyme) 0.771
E (60ng enzyme) 1.020
F (80ng enzyme) 0.817
G (100ng enzyme) 0.853 H (120ng enzyme) 0.827
Protein Expression
Tubes in protein expression were accidentally left in the shaker and were too saturated to proceed. Step one (day one option A) was repeated.
Large culture step:
The OD600 number was monitered until it reached .5 (which sadly never happened)
Measuring OD values - 23uL of total culture
Time: Absorbance at 600nm
10:53 .052
11:32 .064
11:56 .072
1:02 .108 <------ Culture was added until here
1:46 .207
1:55 .350
2:05 .361
2:13 .395
The OD600 value never reached .5 (within 1-2 hours) because it was probably out of the log phase.
Week 13&14 Results and Resubmit to DNA Sequencing
I sent to DNA sequencing twice, first with forward primers: 1:
NNNNN
The results were not a clone (not anything really), but because my master plate did grow and it was evident something was there, so I resubmitted to DNA sequencing, this time with reverse pLIC primers.
The results were not much better, so the move to a surrogate (YopH) was made.
Protein Expression (YopH)
Because Both attempts to get a positive clone from sequencing with PP2C were inconclusive, protein purification was continued with YopH.
The YopH enzyme was transformed into BL21(DE3) cell.
The Small culture+overnight induction (option A) was started: 4 ml LB + 1 colony+ 50 ul Kan were innoculated in a tube, this was repeated for 4 different colonies.
Week 11&12 Cloning Miniprep:
Figure 1: Sample 1
Figure 2: Sample 2
Figure 3: Sample 3
Figure 4: Sample 4
Submitting to DNA Sequencing
Week 9&10
Cloning
Pnic-Bsa4 was successfully cut and the PP2C gene was inserted. Miniprep has not yet been performed and nothing has been sent to sequencing.
Figure 1: Master plate of PP2C in Pnic BSA4
Figure 2: Miniprep of cut pNICBSA4.
*I grew more pNIC-BSA4 and made stock kan this week also*
Week 7&8
MidiPrep
Nanodrop results of the midiprep from our culture of pNICBsa4. The concentration is not so great, and it has some contamination. Cloning will be continued with this, but more pNIC will be grown up in the meantime.
PCR Cleanup
Figure 1: Nanodrop of the PCR2 of our gene PP2C. This has a good concentration with very little contamination. Yay!
Nice images and captions. Try to include a bit of analysis on what you're doing though. Keep it up! - Michael T.
Week 5 & 6
Transformation
Figure 4: Overnight growth of pNIC-BSA4 in LB+Kanamycin media.
Figure 3: pNIC-BSA4 pellets from centrifuging overnight growth.
PCR Squared
Figure 2: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one was skipped, lane two was a 100bp ladder (shown right of image), lane three was sample 1 of PCR squared, lane four was sample 2 of PCR squared, lane five was sample 3 of PCR squared, and lane six was sample 4 of PCR squared. Because this PCR was just amplifying secondary PCR, a bright band would be expected at the gene length (1200bp). All samples showed a band at this length except sample 1.
Secondary PCR
Figure 1: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one is represented with a 100 bp ladder (image on right). Lane 2 is the secondary PCR mixture. A band appeared at the gene length (~1200 bp) so we went on to PCR squared.
Week 3 & 4
Jensen - need your tail primer design info, also use more formalized captions. Include ladder image -- 092713 - Dr. B Primary PCR
Gel produced from run one of primary PCR on a 1x TAE gel (T. Gondii PP2C). Lane 1: skip, lane 2: 100 bp ladder, lane 3: Sample including Primer Mix. A smear appeared as expected in lane 3.
Primer Overlap (Primer Mix)
1 ul of each one of our oligos (30 total) were added to a 1.7 ml tube. 70 ul of autoclaved water was added to this (Total volume=100ul). Primer mix was properly labeled and stored in -20 C feezer.
RE Digest
Gel produced from RE Digest (pGBR22) on 1x TAE Gel. Lane 1: Skip, Lane 2: 1 kb ladder, Lane 3: No RE, Lane 4: No RE, Lane 5: Eco RI, Lane 6: Pvu II, Lane 7:Eco RI and Pvu II. Only lane 6 showed plasmid fragments, meaning samples in lanes 5 and 7 were not cut by the restriction enzyme as they should have been.
Week 1 & 2
PCR with pGBR plasmid
Here you can see the gel result from my PCR run. Sample B appeared nicely at about 900-100bp, sample A had no bands, and sample C had two bands, one at the correct length and another larger one, suggesting too much plasmid was used.
Primer Design
The amino acid sequence of T. Gondii Protein Phosphatase 2C was input into DNAWorks:
Enzyme Assay
Enzyme assay with stock YopH enzyme was performed.
Figure 1: Results of enzyme assay from 234uM sample of YopH enzyme.
Sample Absorbance at 410nm
A (no enzyme) 0
B (10ng enzyme) 0.054
C (20ng enzyme) 0.422
D (40ng enzyme) 0.771
E (60ng enzyme) 1.020
F (80ng enzyme) 0.817
G (100ng enzyme) 0.853
H (120ng enzyme) 0.827
Protein Expression
Tubes in protein expression were accidentally left in the shaker and were too saturated to proceed. Step one (day one option A) was repeated.
Large culture step:
The OD600 number was monitered until it reached .5 (which sadly never happened)
Measuring OD values - 23uL of total culture
Time: Absorbance at 600nm
10:53 .052
11:32 .064
11:56 .072
1:02 .108 <------ Culture was added until here
1:46 .207
1:55 .350
2:05 .361
2:13 .395
The OD600 value never reached .5 (within 1-2 hours) because it was probably out of the log phase.
Week 13&14
Results and Resubmit to DNA Sequencing
I sent to DNA sequencing twice, first with forward primers:
1:
NNNNN
2:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
3:
NNNNN
4:
NNNNN
The results were not a clone (not anything really), but because my master plate did grow and it was evident something was there, so I resubmitted to DNA sequencing, this time with reverse pLIC primers.
1:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNANTNNNNNNNTNNNNNNNGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANTNNNNNNGNNGANANNNNNNCCGCNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNCNNCNCCCNNCCNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNN
2:
NNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNTNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
3:
NNNNN
4:
NNNNN
The results were not much better, so the move to a surrogate (YopH) was made.
Protein Expression (YopH)
Because Both attempts to get a positive clone from sequencing with PP2C were inconclusive, protein purification was continued with YopH.
The YopH enzyme was transformed into BL21(DE3) cell.
The Small culture+overnight induction (option A) was started:
4 ml LB + 1 colony+ 50 ul Kan were innoculated in a tube, this was repeated for 4 different colonies.
Week 11&12
Cloning
Miniprep:
Figure 1: Sample 1
Figure 2: Sample 2
Figure 3: Sample 3
Figure 4: Sample 4
Submitting to DNA Sequencing
Week 9&10
Cloning
Pnic-Bsa4 was successfully cut and the PP2C gene was inserted. Miniprep has not yet been performed and nothing has been sent to sequencing.
Figure 1: Master plate of PP2C in Pnic BSA4
Figure 2: Miniprep of cut pNICBSA4.
*I grew more pNIC-BSA4 and made stock kan this week also*
Week 7&8
MidiPrep
Nanodrop results of the midiprep from our culture of pNICBsa4. The concentration is not so great, and it has some contamination. Cloning will be continued with this, but more pNIC will be grown up in the meantime.
PCR Cleanup
Figure 1: Nanodrop of the PCR2 of our gene PP2C. This has a good concentration with very little contamination. Yay!
Nice images and captions. Try to include a bit of analysis on what you're doing though. Keep it up! - Michael T.
Week 5 & 6
Transformation
Figure 4: Overnight growth of pNIC-BSA4 in LB+Kanamycin media.
Figure 3: pNIC-BSA4 pellets from centrifuging overnight growth.
PCR Squared
Figure 2: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one was skipped, lane two was a 100bp ladder (shown right of image), lane three was sample 1 of PCR squared, lane four was sample 2 of PCR squared, lane five was sample 3 of PCR squared, and lane six was sample 4 of PCR squared. Because this PCR was just amplifying secondary PCR, a bright band would be expected at the gene length (1200bp). All samples showed a band at this length except sample 1.
Secondary PCR
Figure 1: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one is represented with a 100 bp ladder (image on right). Lane 2 is the secondary PCR mixture. A band appeared at the gene length (~1200 bp) so we went on to PCR squared.
Week 3 & 4
Jensen - need your tail primer design info, also use more formalized captions. Include ladder image -- 092713 - Dr. B
Primary PCR
Gel produced from run one of primary PCR on a 1x TAE gel (T. Gondii PP2C). Lane 1: skip, lane 2: 100 bp ladder, lane 3: Sample including Primer Mix. A smear appeared as expected in lane 3.
Primer Overlap (Primer Mix)
1 ul of each one of our oligos (30 total) were added to a 1.7 ml tube. 70 ul of autoclaved water was added to this (Total volume=100ul). Primer mix was properly labeled and stored in -20 C feezer.
RE Digest
Gel produced from RE Digest (pGBR22) on 1x TAE Gel. Lane 1: Skip, Lane 2: 1 kb ladder, Lane 3: No RE, Lane 4: No RE, Lane 5: Eco RI, Lane 6: Pvu II, Lane 7:Eco RI and Pvu II. Only lane 6 showed plasmid fragments, meaning samples in lanes 5 and 7 were not cut by the restriction enzyme as they should have been.
Week 1 & 2
PCR with pGBR plasmid
Here you can see the gel result from my PCR run. Sample B appeared nicely at about 900-100bp, sample A had no bands, and sample C had two bands, one at the correct length and another larger one, suggesting too much plasmid was used.
Primer Design
The amino acid sequence of T. Gondii Protein Phosphatase 2C was input into DNAWorks:
1 MADAKTLLGKVKRATGMGVGEGPSVAKKPKYTATVPGFTPPSGDQRMNEFMAVDTSGEFM
61 RHLYIEEGRTVCASATSRNRRPTSESPHSDDVVVVEGMLRGRPETRVHAMFDGFQGRHSA
121 MWLAQNVMNYLNDLRDVNEEEITRQFERMDGDLRAANLPGGSSALIIFVRYEKKPTEARV
181 VGRQIVPEGAKEFTSVAEALGGPLMPVVAMNFRRDPRAAKGIYTIHVASLGNSRCVLKSG
241 RTAIHLSTPHTASSHKERHRVQAAGGVFTTVNGELLLGGVVPMTRAFGSFDFKKGGQGKL
301 QQDLVSAVPDVTTFFAYPGDDIVAGTAGAFAHFRSHAAIAAAIALYPVSPETVLDAAKAM
361 VVNAKRRKVTKNISTFVRHLPESRTRSQKMLEGTSGENGEX
The following DNA sequence was ouput based on the amino acid sequence:
1 ATGGCGGATGCTAAAACCCTGCTCGGTAAAGTTAAACGTGCGACCGGTATGGGTGTTGGT
61 GAAGGTCCGTCTGTTGCGAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTTACTCCG
121 CCGTCTGGTGACCAGCGTATGAACGAGTTCATGGCAGTTGACACCTCTGGCGAATTCATG
181 CGCCACCTGTACATCGAGGAAGGTCGTACCGTTTGCGCGTCTGCGACTTCTCGTAATCGT
241 CGCCCGACCTCTGAATCTCCGCACTCTGACGATGTTGTTGTGGTTGAGGGTATGCTGCGT
301 GGTCGTCCGGAAACCCGTGTTCACGCGATGTTCGACGGTTTCCAGGGTCGTCACTCTGCG
361 ATGTGGCTGGCGCAGAACGTTATGAACTACCTGAACGACCTGCGCGACGTCAACGAGGAG
421 GAAATTACCCGTCAGTTTGAACGCATGGACGGTGATCTCCGTGCAGCGAACCTGCCGGGT
481 GGCTCTAGCGCCCTGATCATCTTTGTTCGCTACGAAAAGAAACCAACCGAAGCGCGTGTA
541 GTTGGCCGTCAGATCGTCCCGGAGGGTGCGAAGGAATTCACTTCTGTGGCGGAAGCGCTG
601 GGCGGTCCACTGATGCCTGTCGTTGCGATGAACTTCCGTCGTGACCCGCGTGCTGCGAAA
661 GGTATCTACACCATCCACGTAGCGTCTCTGGGTAACTCTCGTTGTGTACTGAAATCTGGC
721 CGTACCGCGATCCACCTCTCTACCCCGCACACTGCCTCTTCTCACAAAGAACGCCACCGC
781 GTACAAGCCGCTGGCGGTGTCTTTACGACGGTAAATGGTGAACTGCTGCTGGGTGGTGTT
841 GTCCCGATGACCCGTGCGTTCGGTTCTTTCGACTTCAAAAAAGGCGGTCAGGGTAAACTG
901 CAGCAGGACCTCGTTTCTGCGGTTCCTGATGTTACCACCTTCTTCGCGTACCCAGGCGAT
961 GACATCGTTGCAGGTACTGCGGGTGCTTTCGCGCACTTTCGTTCTCATGCCGCAATCGCT
1021 GCGGCTATTGCCCTGTATCCGGTTTCTCCTGAGACTGTTCTGGACGCCGCAAAGGCTATG
1081 GTTGTTAACGCGAAACGTCGCAAAGTTACCAAAAACATCTCTACGTTCGTTCGTCACCTC
1141 CCGGAATCTCGTACCCGTTCCCAGAAAATGCTGGAAGGTACTTCCGGTGAAAACGGCGAA
1201 TAA
This was found under the following parameters:
Total Size Of Gene ......... 1203 nt
Protein Residues ........... 401
Mutatable Residues ......... 385
Fixed Nucleotides .......... 48 nt
Degenerate Nucleotides ..... 0 nt
Oligo Size ................. 60 nt
Annealing Temp ............. 62 +/- 1*C
Oligo Concentration ........ 1.00E-7 M
Sodium Concentration ....... 5.00E-2 M
Mg2+ Concentration ......... 2.00E-3 M
Codon Frequency Threshold .. 10%
Repeat Threshold ........... 8 nt
Mispriming Threshold ....... 8/18 (6 exact) nt
From all of this information the following 30 oligonucleotides were formed:
1 ATGGCGGATGCTAAAACCCTGCTCGGTAAAGTTAAACGTGCG 42
2 TTTCGCAACAGACGGACCTTCACCAACACCCATACCGGTCGCACGTTTAACTTTACCGAG 60
3 GGTCCGTCTGTTGCGAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTTACTCCGCCG 60
4 AGGTGTCAACTGCCATGAACTCGTTCATACGCTGGTCACCAGACGGCGGAGTAAAGCCAG 60
5 GTTCATGGCAGTTGACACCTCTGGCGAATTCATGCGCCACCTGTACATCGAGGAAGGTCG 60
6 CGGGCGACGATTACGAGAAGTCGCAGACGCGCAAACGGTACGACCTTCCTCGATGTACAG 60
7 TCTCGTAATCGTCGCCCGACCTCTGAATCTCCGCACTCTGACGATGTTGTTGTGGTTGAG 60
8 CGCGTGAACACGGGTTTCCGGACGACCACGCAGCATACCCTCAACCACAACAACATCGTC 60
9 AAACCCGTGTTCACGCGATGTTCGACGGTTTCCAGGGTCGTCACTCTGCGATGTGGCTGG 60
10 TTGACGTCGCGCAGGTCGTTCAGGTAGTTCATAACGTTCTGCGCCAGCCACATCGCAGAG 60
11 ACCTGCGCGACGTCAACGAGGAGGAAATTACCCGTCAGTTTGAACGCATGGACGGTGATC 60
12 TGATCAGGGCGCTAGAGCCACCCGGCAGGTTCGCTGCACGGAGATCACCGTCCATGCGTT 60
13 GCTCTAGCGCCCTGATCATCTTTGTTCGCTACGAAAAGAAACCAACCGAAGCGCGTGTAG 60
14 AAGTGAATTCCTTCGCACCCTCCGGGACGATCTGACGGCCAACTACACGCGCTTCGGTTG 60
15 GGGTGCGAAGGAATTCACTTCTGTGGCGGAAGCGCTGGGCGGTCCACTGATGCCTGTCGT 60
16 ATACCTTTCGCAGCACGCGGGTCACGACGGAAGTTCATCGCAACGACAGGCATCAGTGGA 60
17 GCGTGCTGCGAAAGGTATCTACACCATCCACGTAGCGTCTCTGGGTAACTCTCGTTGTGT 60
18 CGGGGTAGAGAGGTGGATCGCGGTACGGCCAGATTTCAGTACACAACGAGAGTTACCCAG 60
19 GATCCACCTCTCTACCCCGCACACTGCCTCTTCTCACAAAGAACGCCACCGCGTACAAGC 60
20 CCAGCAGCAGTTCACCATTTACCGTCGTAAAGACACCGCCAGCGGCTTGTACGCGGTGGC 60
21 AATGGTGAACTGCTGCTGGGTGGTGTTGTCCCGATGACCCGTGCGTTCGGTTCTTTCGAC 60
22 ACGAGGTCCTGCTGCAGTTTACCCTGACCGCCTTTTTTGAAGTCGAAAGAACCGAACGCA 60
23 CTGCAGCAGGACCTCGTTTCTGCGGTTCCTGATGTTACCACCTTCTTCGCGTACCCAGGC 60
24 GAAAGTGCGCGAAAGCACCCGCAGTACCTGCAACGATGTCATCGCCTGGGTACGCGAAGA 60
25 GTGCTTTCGCGCACTTTCGTTCTCATGCCGCAATCGCTGCGGCTATTGCCCTGTATCCGG 60
26 AACCATAGCCTTTGCGGCGTCCAGAACAGTCTCAGGAGAAACCGGATACAGGGCAATAGC 60
27 GCCGCAAAGGCTATGGTTGTTAACGCGAAACGTCGCAAAGTTACCAAAAACATCTCTACG 60
28 GAACGGGTACGAGATTCCGGGAGGTGACGAACGAACGTAGAGATGTTTTTGGTAACTTTG 60
29 CGGAATCTCGTACCCGTTCCCAGAAAATGCTGGAAGGTACTTCCGGTGAAAACGGCGAAT 60
30 TTATTCGCCGTTTTCACCGG 20
Nanodrop with pGBR22
Figure 1: The first run of Nano drop to determine the concentration and purity of our pGBR22.
Figure 2: The second run of Nanodrop that confirmed our initial readings of the concentration and purity of the pGBR22.